Discussion - References.
The Western immunoblot, together with the immunohistochemical stainings with MMP-8-specific antibody, demonstrated that MMP-8 (collagenase-2) is present in an inflamed pulp tissue in abundant levels. Most of the MMP-8 in pulpitis eventually originates not only from PMN leucocytes, but also from the other cells, such as macrophage-like cells and plasma cells. In this regard, pulp seems not to differ from other tissues such as periodontium (Kiili et al. 2002) affected by the inflammatory challenge. However, as odontoblasts and pulp fibroblasts can express MMP-8 (Palosaari et al.2000) as wellas other MMPs (Panagakos et al. 1996, Tamura et al. 1996, Llano et al. 1997, Tjaderhane et al. 1998b), the cells of the pulpo-dentinal complex may participate to the matrix remodeling and degradation during the inflammatory processes in the pulp. Indeed, interleukin-1a (IL-1a), tumor necrosis factor-a (TNF-a) and bacterial lipopolysaccharides can stimulate gelatinase expression in pulp fibroblasts in vitro (Panagakos et al. 1996, Tamura et al. 1996, Ueda & Matsushima 2001). Recent studies also show increased expression of collagenase-1 (MMP-1) in cultured human pulp fibroblasts after IL-1a and TNF-a stimulation, whilst prostaglandin E2 (PGE2) stimulates the expression of tissue inhibitor of metalloproteinases- 1 (TIMP-1) (Lin et al. 2001).
Macrophages express various MMPs, including collagenase- 1 (MMP-1), gelatinases (MMP-2, MMP-9), stromelysins (MMP-3, MMP-7, MMP-12) (reviewed by Goetzl et al. 1996) and collagenase-3 (MMP-13) (Imai et al.1998). This may be the first time that macrophage like cells at the site of inflammation have been shown to express MMP-8. The role of collagenases from macrophages may be related to the suggested capability of macrophage-derived MMPs to release growth factors from extracellular matrix to regulate inflammatory reaction (Goetzl et al.1996) or simply to degrade extracellular matrix at the site of inflammation. The immunohistochemical stainings with MMP-8-specific antibody also confirm and further extend the recent findings with the plasma cells present in the close vicinity of bone destructive jaw cysts and periodontitis-affected gingiva, capable of synthesizing MMP-8 (Wahlgren et al. 2001, Kiili et al. 2002).
During the root-canal treatment, a clear decrease in the root-canal exudate MMP-8 levels was observed. The presence of MMP-8 during the second visit, despite the pulp tissue being removed and the canals being cleaned thoroughly during the first appointment, strongly indicates that MMP-8 originated from the periapical inflammation site. This further implies that degradation of the extracellular matrix proteins indicates the active phase of periapical site inflammation to be still present at that time. The virtual absence of MMP-8 in the root-canal exudate during the third visit evidently reflects the disappearance of inflammation and at least partial onset of healing in the periapical environment. This is further supported by the finding that the one specimen failing to show lowMMP-8 level in third appointment was later diagnosed to be vertically fractured. Measuring the presence and levels of MMP-8 from the root-canal exudates during the endodontic treatment could thus serve as a biochemical indicator for monitoring the inflammatory status of the periapical tissue. Therefore, it might be used as a diagnostic guide in deciding the treatment procedures, modalities and medication.
Chlorhexidine, which is sometimes used in root-canal medication, can inhibit MMPs (Gendron et al.1999).Even though the effect of other root-canal medicaments is not known, it would be logical to speculate that Ca(OH)2, because of its high initial pH, may inactivate MMPs which are neutral proteases. However, IFMA measures the amount of total, not the catalytic activity MMP, and therefore, the inhibition of MMP activity by rootcanal medication is not reflected in the results. If the treatment is successful, the number of PMNs in the periapical area will recede and subsequently the MMP-8 levels will be reduced, indicating the success in treatment procedure. Also, the higher proportion of active MMP-8 during the second and third visit (Fig. 2D) indicates that intracanal Ca(OH)2 does not affect the activation of MMP-8 in cases with persistent inflammatory activity, supporting the usefulness of measuring the levels of this surrogate inflammatory mediator from the root canal.
Chair side tests indicating inflammatory activity in periapical lesions have been suggested as future diagnostic tools in endodontics (Takahashi 1998, Metzger 2000). Previous studies have assessed the use of inflammatory mediators such as IL-1b and IL-1a, PGE2 and TNF-a in the evaluation of inflammatory status of periapical lesions (Safavi & Rossomando 1991, Matsuo et al. 1994, Takayama et al.1996, Kuo et al.1998, Ataoglu et al. 2002), but the results have been conflicting. The problem with the above-mentioned inflammatory mediators in this respect may be their multifunctional role in the inflammation, depending on, for example, the concentration, combination and target cells of the mediators. The role of matrix metalloproteinases at the site of the inflammation is, however, clearly defined to the degradation of extracellular proteins (Birkedal-Hansen 1995), offering, therefore, a potential for more specific analysis of inflammatory activity at the periapical site. Therefore, their role in pulpal and periapical pathogenesis should be more thoroughly examined. A chair side MMP-8 test has been recently developed to diagnose and monitor the course and the success of treatment of marginal periodontitis and peri-implantitis (Sorsa et al. 1999, Chen et al. 2000, Ma et al. 2000, Mantylaet al. 2000). Further clinical studies to use MMP measurements from the root canals during the treatment as a diagnostic tool to evaluate the status of the periapical inflammation should be conducted. Finally, in animal experiments specific MMP inhibitors alone or in combinations have decreased oedema and inflammatory tissue damage significantly, suggesting possible therapeutic benefits (Goetzl et al. 1996, Llavernas et al. 2001). There is a wide selection of MMP inhibitors, some of them already in clinical use, suggested as adjunctive therapeutic agents in periodontitis (Sorsa et al.1994, Golub et al.1997, Ciancio &Ashley 1998, Llavernas et al. 2001). MMP inhibition has also been suggested to decrease bone resorption in pathological conditions (Vernillo & Rifkin1998) and dentinal caries progression (Tjaderhane et al. 1998a; 1999, Sulkala et al.2001).MMP inhibition in the root canals and periapical tissues may thus offer new opportunities to rootcanal treatment in the future.
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